Traditionally, monocytes have been viewed as simple, inactive precursors of macrophages. However, after discoveries that blood monocytes encompass several populations, it became clear that monocytes have important independent functions within the vasculature. Nonclassical patrolling monocyte subsets (CX3CR1highLy6C- in mouse and CX3CR1highCD14dimCD16+ in humans) are distinct from the classical monocyte subsets (CCR2highLy6C+ in mouse, and CCR2highCD14+CD16- in humans) and exhibit a unique ability to actively patrol the vascular endothelium under both homeostatic and inflammatory conditions. Although classical monocytes can differentiate into macrophages, it is unclear that nonclassical monocytes undergo this differentiation. Recently, we have found that nonclassical monocytes in circulation phagocytose intravascular tumor cells, thus defining a new and biologically important intravascular function of these monocytes. We hypothesize that a critical function of nonclassical monocytes in circulation is to detect and destroy metastasizing tumor cells in circulation. We discovered that the nuclear receptor Nr4a1 is an essential transcription factor required for the development of nonclassical monocytes. We recently found that Nr4a1-deficient mice that lack nonclassical monocytes have increased tumor seeding in vivo, and very importantly, that transfer of only nonclassical monocytes back into these Nr4a1-deficient mice to restore normal levels of patrolling monocytes in their blood prevents tumor development. Importantly, this function of nonclassical monocytes occurs quite rapidly, within hours of tumor cell infiltration into circulation. We have two specific aims to test our hypothesis Specific Aim 1 will test the hypothesis that nonclassical monocytes direct the killing of tumor cells in circulation and will identify mechanisms for how this occurs. We will test whether Ly6C- nonclassical monocytes kill tumor cells directly or whether they recruit other innate immune cells for killing. We will identify receptor mechanisms for how nonclassical monocytes recognize tumor cells. Specific Aim 2 will test whether increasing nonclassical monocyte numbers in circulation prevents metastasis in vivo. The Nr4a1 agonist Cytosporone B increases nonclassical monocyte numbers in circulation in mice. We will administer Cytosporone B into wild-type mice to increase circulating nonclassical monocyte numbers and will ask whether directly targeting Nr4a1 in monocytes and increasing nonclassical monocyte numbers in circulation prevent tumor cell metastasis. We will also test whether CD14dim monocytes, the human monocyte functional equivalent to Ly6C- nonclassical monocytes, can ingest circulating tumor cells in vivo. These studies will define a critically important homeostatic function for nonclassical monocytes in the vasculature.